Specific antibody to the native form of 2&#39;5&#39;-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2&#39;5&#39;-oligonucleotides in biological systems

ABSTRACT

A new specific antibody to 5&#39;-terminal mono-, di- or triphosphorylated (2&#39;-5&#39;)adenyl-adenoisine oligonucleotides and a method of producing it have been found. The antibody can be used for the quantitative analysis of the oligonucleotides mentioned above in any one of the well known methods of immunological analysis.

This is a continuation of application Ser. No. 672,266, filed Nov. 9,1984.

The invention relates to a specific antibody to the native form of2'5'-oligonucleotides; the method for preparing immunogens by couplingoligonucleotides with 2'5' phosphodiester linkages, instead of thecommon 3'5', and carrying 5' terminal triphosphate to an immunogenicsubstance in such a way that antibodies of extremely high affinity andspecificity can be obtained; and their use as part of highly sensitiveimmuno-assays for 2'5'-oligo(A) in its native 5'-triphosphorylated formor for 5'-terminal mono- or diphosphorylated (2'-5')adenyl-adenosineoligonucleotides, provided the antibody, has an affinity of ¹ /100 ormore of the affinity to 5'-(phospho)₃ (adenylyl 2'-5')₂ adenosine; orfor depleting biological systems of these substances.

Prior art: The oligomeric series of polynucleotides, known collectivelyas 5'-triphospho-(adenylyl 2'-5') adenosine, short name 2'5'-oligo(A),have the structure shown below. ##STR1##

The trimer is shown, and the repeat unit is enclosed in dotted lines.

These compounds have been recently discovered and have aroused a greatdeal of interest because of their association with interferon, andbecaue they represent an entirely novel class of messenger molecules.The literature concerning this subject has been recently reviewed ("TheInterferon Renaissance: Molecular Aspects of Induction and Action".Microbiological Reviews, volume 45, pages 244-266 (1981), by M. Minksand J. Gordon). Most of the inferences concerning the biological actionof 2'5'-oligo(A) have been indirect, and relied on the measurements ofthe enzyme 2'5'-oligo(A) synthetase, which is induced in interferontreated cells. The enzyme can readily be measured as it can be made tofunction very efficiently in cell extracts in the presence of itsactivator, double stranded RNA. Such measurements are of potentialdiagnostic usefulness, since elevated levels are found in a variety ofdiseases, as well as in response to interferon treatment (Lancet ii,8745, pages 497-499, 1981 by A. Schattner, G. Merlin, S. Levin, D.Wallach, T. Hahn and M. Revel and Journal of Interferon Research, volume1, 1981, pages 587-594, by A. Schattner, G. Merlin, D. Wallach, H.Rosenberg, T. Bino, T. Hahn, S. Levin and M. Revel, respectively).Furthermore, a patent application has been filed protecting the use ofsuch enzyme assays (German application DE 30 15 462, by M. Revel, A.Kimchi, L. Schulman and D. Wallach). The usefulness of the assay for2'5'-oligo(A) synthetase is limited by the fact that it is indirect, andthe biological active species is 2'5'-oligo(A) itself. The synthetasedoes not always faithfully reflect the cellular content of2'5'-oligo(A), as can be seen from example number 6 of this application.

Some attempts ave been made to deal with this problem by developingassays for 2'5'-oligo(A) itself. This can be done by measurement, inextracts of cells, of the ability of the 2'5'-oligo(A) to inhibitprotein synthesis or activate an endonuclease (see Methods inEnzymology, volume 79, 1981, pages 199-208, by R. G. Williams, R. E.Brown, C. S. Gilbert, R. R. Golgher, D. H. Wreschner, W. K. Roberts, R.H. Silverman and I. M. Kerr). However, these methods are indirect,cumbersome, not suitable for routine analysis, and involve the use ofunstable reagents. The Group of Kerr therefore developed improvementsincluding a radio-immune and a radio-binding assay (See Methods inEnzymology, volume 79, 1981, pages 216-227, by M. Knight, D. H.Wreschner, R. H. Silverman and I. M. Kerr). Their radio-immune assay wasdependent of the use of an antibody which was insensitive to thepresence of the terminal 5'triphosphate essential for biologicalactivity. Their radio-binding assay was based on the binding of2'5'-oligo(A) to the above nuclease. This assay has been commercializedby Amersham International, using rabbit reticulocyte lysates as thesource of the binding protein. While this assay has a sufficient degreeof specificity, it is of limited usefulness because of the use ofbiochemical preparations which are unstable and need careful handling,because of the presence of degradative enzymes which will preferentiallybreak down the analyte, and because of the finite affinity of thebinding protein, which places a limit on the attainable sensitivity. Allof the above disadvantages are eliminated in the present invention.

In addition to the antibody of the Kerr group mentioned above,monoclonal antibodies have been prepared ("Monoclonal antibodies to5'-triphospho-(2'-5')adenyl adenosine oligonucleotides" in Proceedingsof the National Academy of Sciences of the United States, volume 79,pages 4742-4746, 1982, by H. Cailla, C. LeBorgne de Kauol, D. Roux, M.Delaage and J. Marti), which are also specific for the 2'5' linkage, butnot for the presence of the terminal 5'-triphosphate, in spite of thepromise of the title. The Kerr group used as immunogen (A2'p)₂ A,oxidized with periodate to generate a dialdehyde from the 2' terminalribose, and cross linked this to bovine serum albumin with the aid ofcyanoborohydride. Cailla et al prepared di-succinyl 2'5' ApA and crosslinked this to human serum albumin with the use of carbodiimide.

Since it is clearly preferable to obtain antibodies specific for thebiologically active form of the molecule, it can be concluded thateither these groups had earlier attempted this and failed, or else theydid not attempt to use the native form of 2'5'-oligo(A) with terminal5'triphosphate because they anticipated that it would be degraded in theimmunization procedure.

According to the invention the new antibody may be prepared byparenterally administering to a vertebrate living animal an immunogenconsisting of an immunogenic substance coupled with a fully or partially3'-ribose and 2'-terminal ribose acylated 2'5'-oligo(A), at timeintervals suitable to induce immunization of said animal, gatheringblood which contains the resulting specific antibody, and obtainingantiserum from said blood by clotting and optionally centrifugation, orplasma from said blood by addition of anticoagulant and centrifugation.

It is surprising that the antibody preparations obtained contain only asmall fraction of molecules with the limited specificity reported in theabove two works.

According to the state of the art, nucleotides and oligonucleotides canbe protected from phosphodiesterase attack by reagents which formO-ribose adducts. This is because of the need of the phosphodiesteraseto form a cyclic structure through a 2' hydroxyl (in the case of thecommon 3'-5' linked oligonucleotides) as part of its mechanism, as isshown below: ##STR2## See, for example, "Biochemistry. The chemicalreactions of living cells", by D. E. Metzler. Academic Press, New York,1977, pages, 381-382.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 percent of maximum binding of an antibody as a function of2'5'-oligo(A) added in this competition reaction.

It is surprising that O'ribose modification, which comprises theattachment of the 2'5'-oligo(A) via a linker which promotes itsaccessibility to the immune system of the animal being immunized, alsoprotects the 5'-terminal triphosphate and permits us to raise antibodiesof the specificity revealed in this invention.

The ribose hydroxyls can be acylated with any suitable acidic anhydride,as is well known in the state of the art. See for example the patent ofI. Yamamoto (U.S. Pat. No. 4,350,761, 1982) for the preparation ofadducts with cyclic AMP. Examples of acidic anhydrides which aresuitable for this purpose are e.g. dicarboxylic acid anhydrides.

The preferred form is succinic anhydride, and formula of the resultinge.g fully succinylated 2'5'-oligo(A) is as shown: ##STR3##

Partially succinylated molecules may also be used.

The acyl 2'5'-oligo(A) derivative is then coupled to a suitableimmunogenic substance with a carbodiimide reagent. Suitable immunogenicsubstances are e.g. proteins, peptides, carbohydrates and phospholipids.The carbodiimide reagent can be 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-diethylaminopropyl)-carbodiimide,1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide,1-cyclohexyl-3-(4-diethylaminocyclohexyl)-carbodiimide orN.methyl-N,N'-di-tert-butylcarbodiimidium tetrafluoroborate or othersuitable reagent.

After the coupling reaction e.g. with succinic anhydride, the moleculemay have the structure below, where R represents e.g. an immunogenicprotein coupled via an available amino group: ##STR4## The position ofthe protein is not defined by the reaction conditions, and in principle,any of the succinyl groups could be reacted, or even combinations, whensterically possible. The exact number and positions of the succinylgroups are also not exactly defined by the reaction conditions, andgreat heterogeneity is therefore possible. In spite of this, antibodiesof great homogenity and specificity are obtained by the conditions ofthis invention.

The immunogenic protein may be any suitable protein, such as bovineserum albumin, immunoglobulin of the same or other species as used forthe immunization, or keyhole limpet hemocyanin. The latter is to bepreferred.

Any suitable animal can be used for the immunization, such as sheep,goats, horses, rabbits, rats, mice, chickens, as is commonly used. Ratsyield the best results. The titer of the antibody in the serum isoptimally assayed by the nitrocellulose binding assay described in theexamples, using either [³ H]-2'5'-oligo(A) prepared according to apreviously published procedure ("Synthesis of 2'5'-oligo(A) in extractsof interferon-treated Hela cells", Journal of Biological Chemistry,volume 254, 1979, pages 5058-5064, by M. A. Minks, S. Benvin, P. A.Maroney and C. Baglioni) or [³² P]-pCp-2'5'-oligo(A), labelled by meansof the T4 polynucleotide kinase reaction, and commercially availablefrom Amersham International. The maximum titer is reached after twobooster injections.

The anti-serum so obtained is active at high dilutions (>50,000-fold),is stable to freezing, thawing and lyophilization, and can thus bepackaged, shipped or stored with no difficulty. The serum is alsototally free of any 2'5'-oligo(A) degrading activity, especially at thehigh dilutions used in the assays. This is in contrast to the cellextracts used in the radio-binding assay mentioned above.

The 2'5'-oligo(A) can be extracted from biological material by any ofthe commonly used extraction procedures for making cell-free extracts,provided care is taken to avoid either degradation or re-synthesis underthe extraction conditions. Homogenization with buffered saline may beused, but acid extraction is to be preferred. Optimally, coldtrichloracetic acid extraction is used, preferably in the range 5-20%.The trichloracetic acid can then be removed by ether extraction, orpreferably by freon-octylamine extraction according to a publishedprocedure ("Extraction procedures for use prior to HPLC nucleotideanalysis using microparticle chemically bonded packings", Journal ofChromatographic Science, volume 15, 1977, pages 218-221, by S-C. Chen,P. R. Brown and D. M. Rosie). This procedure is excellent for all kindsof biological materials, including cultured cells, organs, tissues orany biological fluids such as blood, urine, cerebrospinal fluid, etc.The extract may be lyophilized to reduce the volume and removeextraneous volatile material. If desired, an additional step ofchromatography (High Performance Liquid Chromatography or conventionalcolumn chromatography) may be used. Stepwize chromatography ondiethylaminoethyl cellulose is especially simple and useful for removalof the cellular ATP, which may be at concentrations ≧10⁶ -fold theanalyte concentration.

According to the invention the resulting specific antibody is used forthe assay of 5'-terminal mono-, di- or tri-phosphorylated (adenylyl2'-5')_(n) adenosine in biological material by any commonly used methodsof immunological analysis, namely by determination of free or boundligand or determination of free or bound antibody or determination ofantibody-ligand interaction by means of a signal which is modulated bythe antibody-antigen reaction. For instance, this can be done eitherdetermining the reduction of binding of a known highly radioactiveligand, such as [³² P]-pCp-labelled 2'5'-oligo(A), after binding theantibody-ligand complex to a solid support, such as nitrocellulose. Theligand can be labelled with any suitable radioactive isotope. It is alsopossible to label the ligand with a fluorescent, enzymatic orluminescent moiety. The assay can also be of the sandwich type, whereantibody of one specificity is bound to a solid support, used to bindthe unknown analyte, and detected with an antibody of second specificity. Of this pair forming the sandwich, one may be the specific antibodydefined by the present invention, and the other may be of the typedescribed by the groups of Kerr or Cailla described above, or directedagainst the adenosine, as described by Erlanger and Beiser (Proceedingsof the National Academy of the United States, volume 52, 1964, pages68-74). The label need not be in the ligand for any of the above assays,but may be in one of the antibodies, or in a second antibody directedagainst one of the primary antibodies, as is well-known in the art.

The preferred form of assay for detection of fmoles of 5'-terminalmono-, di- or tri-phosphorylated (adenylyl-2'-5')_(n) adenosine inextracts from biological material is the competition binding assayalready mentioned, where the unknown is mixed with a known amount ofknown radioactive ligand, incubated with a dilution of the antibody,mixed with suitable buffer, poured over nitrocellulose in a filtrationapparatus, and the excess unbound ligand removed by washing, andcounting in a scintillation counter. The assay is fast and simple, andthe reaction is complete in a few minutes at reduced temperature. Theassay by reduction of binding of the labelled 2'5'-oligo(A) is as shownhere:

Even more sensitive assays can be constructed with the use of labelledantibodies.

The main application of such assay at present lie in the field ofresearch into the biochemistry of interferon action, and in thebiochemistry of action of hormones, lymphokines growth factors etc. Thewide importance of 2'5'-oligo(A) is evidenced by the fact that2'5'-oligo(A) synthetase is induced in a variety of systems other thaninterferon such as in the differentiating chick oviduct followingestrogen withdrawal, as described ("2-5A synthetase: assay, distributionand variation with growth hormone status", Nature, volume 278, 1979,pages 471-743, by G. R. Stark, W. J. Dower, R. T. Schimke, R. E. Brownand I. M. Kerr), or in Friend Erythroleukemia transformed mouseerythroblasts, triggered into differentiation by various reagents("Increased levels of interferon-induced (2'-5') oligo-isoadenylatesynthetase in mature lymphocytes and in differentiated Frienderythroleukemic cells", Journal of Interferon Research, volume 1, 1981,pages 559-569, by A. Kimchi). Since the real action of the synthetasedepends on its induction and on the simultaneous presence of itsactivator double stranded RNA, such information is of limitedusefulness. In the examples which follow, we demonstrate using themethods of this invention, that indeed, in interferon treated HeLacells, with a high level of 2'5'-oligo(A)-synthetase, the 2'5'-oligo(A)is unaltered from the basal level, while in the differentiating FriendErythroleukemia cells, there is a parallel increase in synthetase and2'5'-oligo(A).

An additional use for the antibody made according to the methods of thepresent invention is as a 5'-terminal mono-, di- or tri-phosphorylated(adenylyl 2'-5')_(n) adenosine chelating agent. It can be added tocell-free protein synthesizing systems, and rescue them from theinhibition by added or endogenous 2'5'-oligo(A). It can also be usedtherapeutically, where it is desired to reduce the cellular levels of2'5'-oligo(A).

Since 2'5'-oligo(A) is such an important newly discovered signalmolecule, it is anticipated that the methods of this invention will leadto the discovery of multitudes of novel uses in research, diagnosis andtherapy.

Diagnostic applications of the 2'5'-oligo(A) synthetase for viralinfection, leukemias, auto-immune and connective tissue diseases havealready been explored by the group of Revel in the publication in Lancet(see above) and for monitoring interferon treatment, in the Journal ofInterferon Research (also mentioned above). Wide applications of assaysfor the biologically active molecule itself, which yield more direct andrelevant information by simpler methodology, can be anticipated in allof the above circumstances.

The more sensitive assays for 2'5'-oligo(A) provided by this inventionalso permits more sensitive assays for the enzyme 2'5'-oligo(A)synthetase. Further, since the enzyme requires double stranded RNA foractivity, this also provides an extraordinarily sensitive assay fordouble stranded RNA. With this method, it is therefore also possible todetermine the small amounts of double stranded RNA in biologicalmaterial which would be necessary for the activation of the2'5'-oligo(A)-synthetase.

In order to explain more fully the invention, the following examples areset forth, it being understood that these examples are not intended tolimit the scope of the invention.

EXAMPLE 1 Preparation of the specific antibody to the native form of2'5'-oligonucleotides

The 2'5'-oligo(A) itself is prepared in quantity using interferontreated Hela cell extracts themselves prepared by published methodology(Minks et al., J. Biol. Chem., 254, 1979, pp 5058-5064). The 500 mlreaction mixture contains 5 mM ATP, 3 mM fructose-1,6-biphosphate, 100mM KOAc, 25 mM Mg(0Ac)₂, 10 mM HEPES/KOH, pH 7.4, 1 mM dithiothreitol,10 μg/ml poly(rI).poly(rC) and 1 mg/ml of post-mitochondrial supernatantfrom interferon treated Hela cells. The mixture is incubated for 15 hrat 30° C., at which stage conversion of ATP to 2'5'-oligo(A) is >30%.The reaction is terminated by boiling for 3 min and denatured proteinsare removed by centrifugation or filtration.

The 2'5'-oligo(A) is separated from ATP and other mononucleotides by ionexchange chromatography on diethylaminoethylcellulose (DEAE cellulose).The clarified boiled extract is diluted with an equal volume ofdistilled water, and loaded on to a DEAE cellulose column equilibratedwith 50 mM NaCl or KCl in 10 mM Tris HCl, pH 7.6, or with 50 mM NH₄ HCO₃or triethylamine bicarbonate pH adjusted to 7.6. The column is loadedwith 100 A₂₆₀ units of the extract per ml of packed column volume.Typically, a 400 ml column is used. The oligonucleotides are eluted witha salt gradient from 50 to 400 mM of the selected salt, using 4 l ofgradient. The ATP elutes first, followed by the 2'5'-oligo(A) oligomersin order of increasing polymeric size. This can also be determined fromthe ultraviolet absorption profile of the column effluent. The desiredfractions are pooled, and the 2'5'-oligo(A) desalted by precipitationwith 5 volumes of acetone, or by lyophilization if the volatile bufferis used.

The 2'5'-oligo(A) is succinylated in a reaction mixture containing 10%(v/v) triethylamine, 50 mg/ml succinic anhydride and 5 mM 2'5'-oligo(A).Typically, 5 ml reaction volume is used. Incubation is at roomtemperature for 10 min. The nucleotides are precipitated with 5 volumesof acetone, followed by de-salting on a column of Sephadex G25.

The succinylated 2'5'-oligo(A) is coupled to keyhole limpet hemocyanin(from Calbiochem) as follows. The hemocyanin is dissolved at 10 mg/ml ina 10 mg/ml solution of succinyl 2'5'-oligo(A), and1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide. HCl is added graduallywhile readjusting the pH to 5.5 with KOH. The condensation reaction isallowed to proceed overnight at room temperature in the dark. Theconjugate is freed from reactants by dialysis against 0.15M NaCl, 0.01Mpotsassium phosphate buffer, pH 6.8 (PBS) for 2 days at 4° C. with atleast 4 buffer changes. The degree of coupling is verified by thespectral alteration before and after coupling; using the A₂₆₀ value as ameasure of the 2'5'-oligo(A) content; or by radioactivity, by inclusionof a small portion of radioactive 2'5'-oligo(A) in the original couplingreaction. The degree of substitution obtained is in the range 80-100moles of oligonucleotide per mole of protein.

Successful immunization of rats, mice or rabbits is obtained with thefollowing procedures. Primary injections are with succinyl-2'5'-oligo(A)conjugate in an equal volume of Complete Freund's Adjuvant and boostersare in an equal volume incomplete Freund's Adjuvant. The conjugate is at1 mg/ml in PBS. Rats are injected intraperitoneally with 0.5 mg ofconjugate and boosted with the same amount at two weekly intervals. Miceare immunized similarly, but with 100 μg per injection. Rabbits areimmunized by subdermal injection of 1 mg of conjugate at five sites onthe back, and boosted with 0.75 mg at three weekly intervals. Rats andmice are bled by the tail vein and rabbits by the ear vein 10 days afterthe previous injection. Rats are preferably 150-200 g females, mice 30 gand rabbits 3 kg.

EXAMPLE 2 Titer of specific antisera

The titers of the antisera are determined from the reciprocal of thedilution which gives 50% retention of the radioactivity to anitrocellulose filter. The antiserum dilution is incubated in 25 μl of20 mM KOAc, 10 mM HEPES-KOH, pH 7.4, 1.5 mM Mg(OAc)₂ containing 1 mg/mlbovine serum albumin (BSA) and 5-6000 cpm of [³² P] terminally labelled2'5'A tetramer pCp (Amersham International: specific activity,approximately 3000 Ci/mmole). After incubation at 0° for 1 hr, themixtures are diluted with 3 ml of ice cold 90 mM KCl, 20 mM Tris-HCl, pH7.6 and passed through 25 mm discs of nitrocellulose (Millipore, typeHAWP, 0.45 μ) with a filtration apparatus. The filters are washed with afurther 2 portions of the same buffer, and retained radioactivitycounted by Cerenkov radiation in a scintillation counter. Maximal titersof 50,000, 5,000 and 1,000 are usually obtained for rats, mice andrabbits, respectively, and after the second booster injection.

EXAMPLE 3 Competitive binding assays

In the simple radio-immune assay described in Example 2, the unknown isdetermined by dilution of the radioactivity bound from the constantamount of known radioactive probe. Other related substances will bind tothe antibody more or less well, depending on their relative affinities,and may thus also give a reduction in the bound radioactivity. Thisinhibition is a measure of the affinity of the related compound for theantibody, and will also indicate to what extent such compounds mightinterfere with the assay, if present in the material being analysed. Theconcetration of the related substance giving 50% inhibition of thebinding of the radioactive probe is used as a measure of its relativeaffinity. A collection of 2'5'-adenylates and related compounds wereprepared as follows. The dimer, trimer, tetramer and pentamer wereobtained from the DEAE-cellulose fractionation of example 1. Thetetramer was partially digested with bacterial alkaline phosphatase andthe products separated by high performance liquid chromatography on aPartisil SAX column (Whatman), with 10 mM potassium phosphate buffer, pH6.8, 20% ethanol and a gradient from 10 to 500 mM KCl. Acetyl andsuccinyl derivatives of the tetramer were obtained by reaction withacetic and succinic anhydride, according to the method of Example 1.Other compounds were from commercial sources. The amounts of the variouscompounds which gave 50% inhibition of the maximal binding of the [³²P]-labelled probe (see Example 2) are listed below.

    ______________________________________                                                               fmoles                                                 ______________________________________                                        pppA(2'p5'A).sub.4           2.5                                              pppA(2'p5'A).sub.3           2.5                                              pppA(2'p5'A).sub.2           2.5                                              pppA(2'p5'A)                 850                                              2-O--succinyl pppA (2'p5'A).sub.3                                                                          2,0                                              2-O---acetyl pppA(2'p5'A).sub.3                                                                            2.5                                              ppA(2'p5'A).sub.3            12                                               pA(2'p5'A).sub.3             25                                               A(2'p5'A).sub.3              500                                              A(2'p5'A).sub.2              500                                              A(3'p5'A).sub.2              2,500,000                                        pA(3'p5'A).sub.3             250,000                                           AMP, ADP, ATP, GTP                                                                                        >3,000,000                                       CTP, UTP, NADH, pCp                                                           ______________________________________                                    

The assay is therefore able to detect fmole amounts of oligonucleotideswith appropriate specificity. The affinity of the antibody is so highthat the same results will be obtained with any reaction volume betweenthe 25 μl specified in the preceeding example and 1 ml. The affinity ofthe antibody is ˜10¹² M⁻¹. The affinity of the antibody for the5'-phosphorylated forms of 2'5'-oligo(A) is 20-200 times that for thede-phosphorylated core. This supports the concept that the antibody isspecific for 5'-phosphorylated 2'5'-oligo(A).

EXAMPLE 4

Preparation of extracts for 2'5'oligo(A) radioimmunoassays

This is an example for cells growing in culture, but is universallyapplicable to any biological material: cell cultures, organs, tissues,bodily fluids such as blood, urine, cerebrospinal fluids, milk or anyother suitably available material. Cells in a petri dish are rapidlywashed with buffered saline (this step is dispensed with in the case ofbodily fluids), then enough ice cold 5% trichloracetic acid (TCA) tocover the layer of cells (3 ml for a 7 cm diameter petri dish) and thedish is kept on ice for 5 min. The TCA and detached cells are removedand centrifuged to remove the insoluble material, and the dish andpellet are re-extracted with a similar volume of 5% TCA and thesupernatants pooled. Alternatively, in the case of a cell suspensionculture, the cells are pelleted by centrifugation at room temperature,the cells resuspended in a volume of buffered saline equal to the volumeof the pellet, and approximately 10 volumes of 5% TCA are added. Theinsoluble material is removed by centrifugation and the pellet extractedwith 1/2 the previous volume of 5% TCA. The supernatants are pooled. Alloperations with TCA are performed between 0° C. and 5° C. The pooledsupernatants are freed of TCA by extraction with an equal volume of 30%(v/v) tri-N-octylamine in Freon 113 (Dow Chemicals). The upper aqueousphase is collected and the Freon phase re-extracted with 1/2 thepreceeding volume of distilled water. Since biological material maycontain ATP in the mM range, and it is desirable to measure2'5'-oligo(A) in the nM range, it may be desirable to remove such 10⁶-fold excess of ATP, which might interfere with the assay. The ATP andother nucleotides which might interfere with the assay may then beremoved chromatographically with DEAE cellulose (Whatman DE-52)equilibrated with 125 mM NH₄ HCO₃ (pH 7.8). The loading ratio is thesame as for the preparative columns of example 1, but microcolumns of200 μl are optimal for this analytical scale. The column is washed with15 ml of the same buffer to remove the ATP and other unbound material.The 2'5'-oligo(A) is eluted with 2 ml of 0.5M NH₄ HCO₃, and the samplesare lyophilized after addition of 1/10th volume of ethanol. The samplesmay be stored indefinitely in anhydrous form and re-dissolved withsuitable volumes of solvent for the radio-immuno assay as described inexample 3.

EXAMPLE 5 Determination of 2'5'oligo(A) levels in differentiating cells

Friend mouse erythroleukemia cells can be induced to differentiate andbecome erythropoietic under the influence of a variety of reagents,especially dimethylsulfoxide. Such cells are also known to produce theenzyme 2'5'-oligo(A) synthetase. Before the method of the presentinvention, it was not possible to determine the 2'5'oligo(A) directly inthese cells. The determination is described in this example. The Friendcells (clone FBU or N46AP) are grown in Dulbecco's modified Eagle'sMedium containing 10% fetal calf serum, 2% (v/v) dimethylsulfoxide forFBU or 1.4% (v/v) DMSO for N46 AP and 1 mM glutamine. The cells arediluted with the same medium daily to maintain a density of 5.10⁵ cellsper ml. At various times, 30 ml cultures are collected and processed asdescribed in the preceeding example. The lyophilized material isdissolved in the buffer corresponding to the reaction mixture for theassay, as in example 2, but containing 0.1 mg/ml of bovine serumalbumin. The 2'5'-oligo(A) content is determined from the reduction ofthe binding of the [³² P] labelled probe as in example 2 and 3. Thefollowing results are obtained:

    ______________________________________                                                           2'5'-oligo(A)                                              Cell line Days post-DMSO                                                                         (fmoles/10.sup.6 cells)                                    ______________________________________                                        FBU          0         2.6                                                                 1         28                                                                  2         28                                                                  4         20                                                     N46AP        0         7.5                                                                 1         7.8                                                                 2         4.3                                                                 3         17.5                                                                5         63                                                                  6         53                                                     ______________________________________                                    

In the case of the N46AP cell line, there is a delay between the firstappearance of the 2'5'-oligo(A) snthetase and the 2'5'-oligo(A) itself,while in the FBU line the two go in parallel.

This example demonstrates for the first time the possibility ofdetermination of 2'5'-oligo(A) in cells not treated with interferon.

EXAMPLE 6 Determination of 2'5'-oligo(A) in interferon treated Helacells

Cultures of Hela cells respond very well to interferon in the inductionof 2'5'-oligo(A) synthetase (see example 1). The basal 2'5'-oligo(A)itself was too low to be measured by other methodology. This exampledemonstrates such measurement. Hela cells of the S3 line are cultivatedin the presence of 20 units/ml of interferon α2 for 24 hrs. The cellsare centrifuged and processed according to example 4. The 2'5'-oligo(A)synthetase is assayed by methods well described in the literature inparallel cultures. The synthetase increases 10-fold over the basallevel, while the 2'5'-oligo(A) itself remains at the basal level of0.45±0.15 nM. This shows that 2'5'-oligo(A) synthetase levels are notinvariably indicative of the biologically active species, 2'5'-oligo(A).

EXAMPLE 7 Enhanced sensitivity assay for 2'5'-oligo(A) synthetase

Because of the extremely efficient reaction condition of cell-freesystems, it is possible to assay low levels of 2'5'-oligo(A) synthetase.However, it is sometimes desirable to measure low levels from extremelysmall amounts of precious starting material. By coupling the extremelysensitive measurement of 2'5'-oligo(A) according to the methods of thepresent invention, with the enzymatic assay for 2'5'-oligo(A)polymerase, the enzyme can be determined with 10⁵ -fold highersensitivity than was previously possible. In the example, Swiss mouse3T3 fibroblast cells are stimulated into a cell cycle by a variety ofhormones and factors (for literature on this subject see the article"Criteria for the establishment of the biological significance ofribosomal protein phosphorylation" in Current Topics in CellularRegulation, volume 21, 1982, pages 89-99, by J. Gordon, P. J. Nielsen,K. L. Manchester, H. Towbin, L. Jimenez de Asua and G. Thomas). Qiescent3T3 cells in Dulbecco's modified Eagle's minimal medium and 10% fetalcalf serum, in 10 cm Petri dishes are treated overnight with variousreagents. On the following day the monolayers are washed with phosphatebuffered physiological saline and the cells suspended in 2 ml per dishof the same. The cells are centrifuged at 800 g for 5 min and the packedcells treated with twice the pellet volume of 10 mM HEPES-KOH, pH 7.4,20 mM KOAc, 1.5 mM Mg(OAc)₂, 2 mM DTT, 0.1% (v/v) triton X-100, and 10%glycerol, for 10 min at 0°. The cell debris are removed bycentrifugation at 30,000 g for 10 min and the supernatant is used as thesource of 2'5'-oligo(A) polymerase. The supernatant (40 μl, 30 A₂₆₀) ismixed with 100 μl of poly(rI).poly(rC)-agarose beads (P-L. Biochemicals)and 100 μl of 20 mM Tris-HCl pH 7.6, 225 mM KOAc, 12 mM Mg(OAc)₂, 1 mMdithiothreitol, 10 μM phenylmethylsulfonylchloride and 10 % glycerol.The non-adsorbed material is removed after centrifugation to pellet thebeads and the agarose is washed three times with 200 μl aliquots of thelatter buffer. The 2'5'-oligo(A) synthesis is allowed to proceedovernight in the presence of 50 μl of the latter buffer supplementedwith 10 mM ATP containing 500,000 cpm of [³ H]-ATP. The reaction isterminated by addition of 150 μl of 90 mM KCl, 20 mM Tris-HCl pH 7.6.The beads are removed by centrifugation and the supernatant divided into2 portions, one for a conventional assay and the second for theradio-immune assay of the reaction product. One portion (198 μl) isanalysed for conversion of the ATP to 2'5'-oligo(A) from the retentionof radioactivity on an analytical DEAE cellulose column (see example 4).The other portion (2 μl) is diluted in the range 250 to 10,000-fold andthe 2'5'-oligo(A) determined by the radioimmune assay (see example 3).The following results are obtained.

    ______________________________________                                                   [.sup.3 H]--ATP→[.sup.3 H]--2'5'-                                                      Radio-immune assay                                            oligo(A)        for 2'5'-oligo(A),                                 Treatment  cpm/assay       pmoles/assay                                       ______________________________________                                        None       556              50                                                +10 μM prosta-                                                                        632             100                                                glandin F2α                                                             +100 u/ml inter-                                                                         4109            2000                                               feron                                                                         Background of                                                                            (197)            0                                                 assay                                                                         ______________________________________                                    

With radioactive ATP, more than 10⁷ cells must be extracted to obtain asignificant signal. The sensitivity is limited by the high Km of theenzyme (c. 1 mM) and the background of the assays itself. In general, itis difficult to discriminate lower than 1/10⁴ of the input ATP. Theantibody assay improves both the sensitivity and the selectivity. Thediscrimination factor between 2'5'-oligo(A) and ATP is ca. 10⁶ -fold,and this can be enhanced to 10⁸ -fold with a DEAE cellulose column step.The minimal amount of 2'5'-oligo(A) to give a signal is in the fmolerange, considerably lower than that needed to detect the 2'5'-oligo(A)in the above table. The enhanced sensitivity of the radio-immune assayis thus 10⁵ -fold. It thus permits the use of smaller amounts ofbiological material, biological material with very low activity, or themeasurement of smaller amounts of material which activitates thesynthetase, than would otherwise be possible.

EXAMPLE 8 Non-isotopic immuno-assays for 2'5'-oligo(A)

Great developments have been made in recent years in easier and saferalternatives to radio-immunoassay techniques. For example, such methodshave been developed which are of great simplicity and sensitivity, basedon nitrocellulose as a solid support and peroxidase-conjugatedantibodies as label (see "A dot-immunobinding assay for monoclonal andother antibodies", Analytical Biochemistry, volume 119, 1982, pages142-147, by R. Hawkes, E. Niday and J. Gordon and U.K. patentapplication No. 2099578 "New Kits for Immunological Analysis", 1981, byJ. Gordon, R. Hawkes, E. Niday and H. Towbin). Conjugated 2'5'-oligo(A)is prepared with bovine serum albumin instead of hemocyanin according toexample (1). This is diluted to 0.2 mg/ml in TBS (0.01M Tris-Cl, pH 7.6,0.15M NaCl), 1 mg/ml BSA. Rat anti-serum against 2'5'-oligo(A) preparedaccording to example (2) is diluted 1:10,000 in TBS, 1 mg/ml BSA, 0.01%Nonidet P40 (Shell Chemicals). Standard and unknown samples are dilutedinto 1 ml portions of the diluted anti-serum, and incubated for 1 hr atroom temperature. These are then added to the nitrocellulose andincubated for a further 2 hrs at room temperature, with gentleagitation. The strips are then washed with TBS and incubated a further 2hrs in 1 ml of peroxidase conjugated rabbit anti-rat immunoglobulins(DAKO, Copenhagen), diluted 1:500 in TBS, 1 mg/ml BSA, 0.01% NonidetP40. The strps are then washed again with TBS and incubated in 1 ml 0.6mg/ml chlornaphthol and 0.01% H₂ O₂ in TBS for 15 min. The inhibition ofthe color reaction is then taken as a measure of the amount of unknown2'5'-oligo(A). A reduction of 50% is obtained with 100 fmoles of2'5'-oligo(A).

EXAMPLE 9 Kits for assay of 2'5'-oligo(A)

Such kits may consist of two parts: (a) for the extraction and (b) forthe performance of the assay itself. These are provided either togetheror separately.

(a) comprises materials for carrying out the procedure of example (4) ina stable, packaged form. Trichloracetic acid is provided in packscontaining 5 g, to be made up to 100 ml with distilled water.Physiological saline pH 6.8 is provided in packs containing 0.88 g NaCl,0.14 g KH₂ PO₄ and 0.23 g K₂ HPO₄.3H₂ O, to be made up to 100 ml withdistilled water. Freon 113 and tri-N-octylamine are provided in ampoulesof 35 and 15 ml, respectively, which, when mixed, provide the non-aqeousphase for the extraction of the trichloracetic acid. The provision ofthe latter two ingredients as part of a kit is especially useful as theyare not generally available in biochemical or clinical laboratories. Thelatter volumes provide enough for approximately 50 assays. Disposablemicrocolumns are made by packing 0.2 ml of DEAE cellulose (Whatman DE52)equilibrated with 125 mM NH₄ HCO₃ and 0.05% NaN₃ as preservative, indisposable polypropylene 2 ml Econocolumns (from Biorad Corporation).These are packed in the kit with the top sealed with the stopperprovided and the bottom with a luer fitting. NH₄ HCO₃ is provided inpacks of 9.88 g, which can be made up to 1 liter for the column washbuffer or to 250 ml for the elution buffer.

(b) For a radio-immune assay, the kit contains 2'5'A tetramertriphosphate [³² P]pCp 3' end labelled, 25μ Ci, as supplied by AmershamInternational, rat antiserum against 2'5'-oligo(A) prepared as inexample (1). The antiserum is diluted 50,000 fold in 20 mM KOAc, 10 mMHEPES-KOH, pH 7.4, 1.5 mM Mg(OAc)₂, 1 mg/ml bovine serum albumin,divided into 1.25 ml portions and lyophilized. Each portion providesmaterial for 50 assays following reconstitution. Washing buffer is madefrom portions of 0.67 g KCl, 0.24 g Tris and 1.9 ml 1N HCl. The mixtureis lyophilized and packed. Each pack provides 100 ml of wash buffer whenreconstituted with distilled water. Alternatively, the probe may be madeby conjugation of succinyl 2'5'-oligo(A) with tyrosine by methods knownfor cyclic AMP (see Cailla, H. L. & Delaage, M. A. in AnalyticalBiochemistry 48 (1972), page 62), and labelled with [¹²⁵ I] by thechloramine T method.

The kit may also be assembled for enzyme-based immuno-assay. In itssimplest form, multi-well dishes are provided with 3 mm×3 mm squares ofnitrocellulose (Millipore type HAWG) in each well. The squares areprepared with 0.5 μl of 1:50 dilute antiserum and saturated with 10%horse serum as in example (8). The remaining components of the kit areas described in U.K. patent application No. 2099758.

We claim:
 1. A process of producing antibody specific for 5'-terminalmono-, di- or tri-phosphorylated (2'-5') adenyl-adenosineoligonucleotides which comprises parenterally administering to avertebrate living animal an immunogen consisting of an immunogenicsubstance coupled with a fully or partially 3'-ribose and 2'-terminalribose acylated 5'-terminal mono- di- or tri-phosphorylated (2'-5')adenyl-adenosine oligonucleotide at time intervals suitable to induceimmunization of said animal, gathering blood which contains theresulting specific antibody, and obtaining serum from said blood byclotting and optionally centrifugation or plasma from said blood byaddition of anticoagulant and centrifugation.
 2. A process according toclaim 1 in which the immunogenic substance may be a protein, a peptide,carbohydrate or phospholipid.
 3. A process according to claim 1 in whichsaid acylated (2'-5') adenyl-adenosine ologonucleotide is acylated withdicarboxylic acid anhydrides.
 4. A process according to claim 2 in whichsaid acylated (2'-5') adenyl-adenosine oligonucleotide is acylated withdicarboxylic acid anhydrides.
 5. A process according to claim 1 in whichthe immunogenic substance is coupled to any one or all of the acylatedpositions.
 6. A process according to claim 5 in which the immunogenicsubstance may be a protein, peptide, carbohydrate or phospholipid.
 7. Aprocess according to claim 5 in which said acylated 2'-5')adenyl-adenosine oligonucleotide is acylated with dicarboxylic acidanhydrides.
 8. A process according to claim 6 in which said acylated(2'-5') adenyl-adenosine oligonucleotide is acylated with dicarboxylicacid anhydrides.